Revealing the hidden diversity of Gyrodactylus communities (Monogenea, Gyrodactylidae) from Nearctic Catostomidae and Leuciscidae fish hosts (Teleostei, Cypriniformes), with descriptions of ten new species

Despite the high diversity of freshwater fishes in the Nearctic region, little is known about the composition of their parasite communities. We addressed the diversity of viviparous monogeneans of Gyrodactylus parasitizing highly diversified cypriniform fish inhabiting Nearctic watersheds. Nowadays, a thorough assessment of Gyrodactylus spp. diversity requires both morphological traits and genetic data. A combination of taxonomically important haptoral features and sequences of the ITS regions and 18S rDNA revealed 25 Gyrodactylus spp. parasitizing two catostomid and 15 leuciscid species sampled in six distinct localities in the United States and Canada. These include ten Gyrodactylus species recognized as new to science and described herein (G. ellae n. sp., G. hamdii n. sp., G. hanseni n. sp., G. huyseae n. sp., G. kuchtai n. sp., G. lummei n. sp., G. mendeli n. sp., G. prikrylovae n. sp., G. scholzi n. sp., and G. steineri n. sp.), seven already known species, and finally eight undescribed species. Overall, Nearctic Gyrodactylus spp. exhibited haptoral morphotypes known from fish hosts worldwide and those apparently restricted to Nearctic Gyrodactylus lineages like the typical ventral bar with a median knob and a plate-like membrane, or the additional filament attached to the handles of marginal hooks. The integrative approach further evidenced possible ongoing gene flow, host-switching in generalist Gyrodactylus spp., and regional translocation of monogenean fauna through fish introductions. The study highlights the hitherto underexplored morphological and genetic diversity of viviparous monogeneans throughout the Nearctic region.


Introduction
The North American continent hosts one of the most diverse temperate freshwater fish faunas in the world, with several thousand described and numerous undescribed species [28,56].With a wide geographical distribution, cypriniforms compose the most diverse monophyletic freshwater fish clades [63,73], counting over 4000 species [28].Cyprinoidei, the most speciose lineage of cypriniforms, comprises Cyprinidae (carps and minnows) and Leuciscidae (true minnows) as the largest and most diverse groups [92].Overall, over 80 genera were established for leuciscids [28], distributed in Nearctic and Palearctic Eurasia [6,64].Leuciscidae is a single cyprinoid family naturally distributed in North America.Cypriniformes in the Holarctic region are also represented by suckers (Catostomidae, Catostomoidei), with 13 catostomid genera native to North America and a single genus native to Asia [38,100,101].
Monogeneans are highly host-specific parasites [105], reflecting the distribution of their hosts across continents [51,94].Gyrodactylus von Nordmann, 1832 (Gyrodactylidae Cobbold, 1864) is a well-known, highly diverse monogenean genus with almost 500 known species parasitizing teleost fish [1,8], including some highly pathogenic species [2].While life history traits of most Gyrodactylus flatworms predominantly remain unknown, Gyrodactylus spp.have been recognized to parasitize representatives of almost 20 bony fish orders and exhibit a variable degree of host specificity [3,51,80,105].This might be linked to the direct life cycle and the lack of a specialized transmission stage, which favors host switching, in contrast to limited host choices that face the larval stage (oncomiracidia) of oviparous monogeneans [45].Members of Gyrodactylus are known for their site specificity: they are present on external surfaces like skin and fins (for instance G. atratuli Putz & Hoffman, 1963 [84]), restricted to the gills only (for instance G. baeacanthus Wellborn & Rogers, 1967 [103]), or present on the skin, fins and gills as well (for instance G. corleonis Paladini, Cable Fioravanti, Faria & Shinn, 2010 [78]).
In general, the description of any monogenean species based on morphological characters alone can be problematic and requires considerable expertise.Morphologically, Gyrodactylus spp.show inconspicuous diversity with relatively little variations in their attachment apparatus, termed the haptor.Although Malmberg [59] elaborated a morphological method of Gyrodactylus classification based on the excretory system, the discrimination of gyrodactylid taxa remains problematic.Malmberg's "species-group" concept was for a long time regarded as the miracle approach for separating species, but this view was later challenged when genetic data recovered the G. wageneri group as paraphyletic [9], while morphology and host preference suggested monophyly [3].In addition, sclerotized haptoral features in Gyrodactylus (anchors, transverse bars and marginal hooks) may vary ecophenotypically depending on parasite age, season, geographic distribution, location on host, and host species (see, for instance, [25,26]).
The integration of methods other than genetics for discriminating Gyrodactylus spp.has not always been successful.This was the case, for instance, with the application of statistical classifiers on high-quality scanning electron micrographs obtained from G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, two well-known pathogenic species from salmonids [93].On the contrary, the combination of traditional morphological characterizations and DNA sequences has been shown to be efficient to a certain degree in Gyrodactylus spp.delineation (see, for instance, [43,61]).However, in the case of G. salaris and G. thymalli, almost no genetic variation was observed using the internal transcribed spacer (ITS; ITS1-5.8S-ITS2)regions of rDNA [16,109], whereas, later, these two species were shown to be conspecific with microRNA loci analyses [30].The ITS fragments evidenced variations between G. salaris, G. derjavini Mikailov, 1975, and G. truttae Gläser, 1974 parasitizing salmonids [16].A few other genetic markers, such as the ribosomal intergenic spacer (IGS) and cytochrome c oxidase subunit I (COI), were shown to be useful in terms of revealing genetic variation compared to ITS sequences [17,[34][35][36]67].
Gyrodactylus spp.have a worldwide distribution in freshwater, brackish, and marine habitats [4], and mostly parasitize cypriniform fishes [1,37].Gyrodactylus, with more than 50 currently known species, represents the second largest monogenean genus known from Nearctic fishes.Leuciscids in Palearctic and Nearctic regions harbor different species of Gyrodactylus (except for a few co-introduced species in North America) [51].
In recent decades, research targeting the parasite fauna in Nearctic freshwater fishes has lagged behind similar research in Europe [91].While North America possesses a higher diversity of cypriniform fishes than Europe, the known parasite species richness, specifically that of monogeneans and tapeworms per cypriniform species in Europe, is much higher compared to North America [51].
In light of the lack of knowledge on current fish parasite diversity [51], our study was specifically focused on viviparous monogeneans of Nearctic cypriniform fish fauna with the aim of recovering the hidden diversity of Gyrodactylus communities in broadly diversified Leuciscidae and Catostomidae.We applied an integrative approach to combine morphological characters and molecular markers.

Fish host collection and identification
Cypriniform fish hosts were collected in 2018, 2019, and 2022 from distinct freshwater systems in the United States (Arkansas, New York, Mississippi, and Wisconsin) and Canada (Quebec).Information related to cypriniform fish hosts, their sampling localities, and Gyrodactylus diversity is shown in Table 1.Fish identification was performed by local collaborators (listed in acknowledgements) or based on common identification keys.Fieldwork was carried out with the approval of the official local authorities (provided to US partners).
The identity of the investigated cypriniform hosts was further checked by means of molecular barcoding using the partial cytochrome b (cyt-b) gene.Mitochondrial DNA of host species was isolated from fin clips preserved in 96% ethanol using a DNeasy Ò Blood & Tissue Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions.Amplification of the cyt-b gene was performed using forward primer GluF (5 0 -AACCACCGTTGTATTCAACTACAA-3 0 ) and reverse primer ThrR (5 0 -ACCTCCGATCTTCGGATTACAAGACC-G-3 0 ) [57].PCR reactions consisted of 1 U of Taq polymerase (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA), 1 Â PCR buffer, 1.5 mM MgCl 2 , 0.4 mM of each dNTP, 0.4 lM of each primer, and an aliquot of 30 ng (1 lL) of genomic DNA in a total volume of 25 lL.PCR was carried out in a Mastercycler ep gradient S (Eppendorf AG, Hamburg, Germany) with the following steps: 2 min at 94 °C followed by 39 cycles of 45 s at 92 °C, 90 s at 48 °C, and 105 s at 72 °C, and 7 min of final elongation at 72 °C.The PCR product was purified by ExoSAP-IT™ (Amplia, Bratislava, Slovakia) and was sequenced directly in both directions using the same primers as in the amplification reaction.The initial amplification was carried out using a BigDye Ò Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA) and an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems).Raw nucleotide sequences were edited using Sequencher software v. 5.0 (Gene Codes, Ann Arbor, MI, USA) and aligned using ClustalW [98] as implemented in MEGA v. 11 [97].The identification of cypriniform species based on a sequence similarity approach was carried out using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi:blastn, default settings).Newly generated sequences for the cypriniform species were deposited in GenBank (see the species descriptions below).Catostomid and leuciscid fish host nomenclature follows FishBase [29].

Parasite collection and morphometric study
During the field trip, fins and gills were examined for Gyrodactylus spp.using an MST130 stereoscopic microscope.Monogenean specimens were removed using surgical needles and mounted on slides with a mixture of glycerine and ammonium picrate (GAP) [58].Selected specimens of each collected monogenean species were cut in half using fine needles under a dissecting microscope.The anterior part of the body with male copulatory organ (MCO) was placed in a 1.5 mL Eppendorf tube with 96% ethanol for DNA extraction, while the posterior part with haptoral sclerites (anchors, bars and marginal hooks) was fixed in GAP for morphological characterization.Gyrodactylus spp.were identified using original descriptions (see the result sections for references).Measurements and photographs were taken using an Olympus BX51 phase-contrast microscope and Olympus Stream Image Analysis v. 1.9.3 software (Olympus, Tokyo, Japan).Measurements of Gyrodactylus spp.are shown in micrometers and are given as the mean followed by the range and the number of measurements (n) in parentheses.Drawings of the haptoral sclerotized parts were made on flattened specimens using an Olympus BX51 microscope equipped with a drawing tube and edited with a graphic tablet compatible with Adobe Illustrator CS6 v. 16.0.0and Adobe Photoshop v. 13.0 (Adobe Systems Inc., San Jose, CA, USA).Infection indices were calculated for all collected Gyrodactylus spp. with a sufficient sample size (sample size for a few non-described species was very low, see below) according to [7].The type-material was deposited in the National Museum of Natural History (MNHN, Paris, France) under accession numbers HEL1996-HEL2034.

Genetic characterization
Each Gyrodactylus specimen preserved in 96% ethanol was dried using an Eppendorf 5301 Concentrator.Total genomic DNA was extracted using a DNeasy Ò Blood & Tissue Kit following the protocol for the purification of total DNA from animal tissues.Two nuclear ribosomal DNA markers suitable for the differentiation of Gyrodactylus spp.were used (for instance, [9,31,61,79,111]).A fragment spanning ITS1, 5.8S and ITS2 (ITS regions) was amplified using forward primer ITS1F (5 0 -GTTTCCGTAGGTGAACCT -3 0 ) [88], complementary to the sequence at the 3 0 end of the 18S rRNA gene, and reverse primer ITS2 (5 0 -TCCTCCGCTTAGTGATA-3 0 ), complementary to the sequence at the 5 0 end of the 28S rRNA gene [16].A partial fragment of 18S rDNA containing the V4 region, which exhibits intraspecific variation in Gyrodactylus [15,61], was amplified using the primer pairs PBS18SF (5 0 -CGCGCAACT-TACCCACTCTC-3 0 ) and PBS18SR (5 0 -ATTCCATGCAA-GACTTTTCAGGC-3 0 ) [13].Polymerase chain reactions (PCRs) for the 18S rDNA gene and ITS region were performed in a final volume of 30 lL, containing 1xPCR buffer (Fermentas), 1.5 mM MgCl 2 , 200 lM of each dNTP, 0.5 lM of each primer, 1 U of Taq Polymerase (Fermentas) and 5 lL of template DNA.The PCRs were carried out in the Mastercycler ep gradient S (Eppendorf) using the following steps: i) ITS regions: an initial denaturation at 96 °C for 3 min, followed by 39 cycles of denaturation at 95 °C for 50 s, annealing at 52 °C for 50 s and an extension at 72 °C for 50 s, and a final elongation at 72 °C for 7 min; and ii) 18S region: an initial denaturation at 95 °C for 3 min, followed by 39 cycles of denaturation at 94 °C for 1 min, annealing at 54 °C for 45 s and an extension at 72 °C for 1 min 30 s, and a final elongation at 72 °C for 7 min.PCR products were electrophoresed on 1.5% agarose gels strained with Good View (SBS Genetech, Bratislava, Slovakia) and then purified using ExoSAP-IT™ (Amplia, Bratislava, Slovakia), following the manufacturer's protocol.The purified PCR products were sequenced directly in both directions using the PCR primers.For sequencing of the ITS regions, one additional internal primer, ITSR3A (5 0 -GAGCCGAGT-GATCCACC-3 0 ) [61], was used.Sanger sequencing was carried out using a BigDye Ò Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems).Obtained DNA sequences were assembled and edited using Sequencher software.Newly generated sequences for Gyrodactylus spp.were checked by the nBLAST Search Tool to assess any similarity to available congeners, then deposited in GenBank (see the species descriptions below).
The genetic variation among newly generated sequences of Gyrodactylus spp. was evaluated using MEGA [97].Sequences of the 18S rDNA and ITS regions from several Eurasian Gyrodactylus representatives that were shown to be genetically closely related to our studied species were retrieved from the GenBank database to assess the genetic variations.This was estimated using uncorrected genetic p-distances in MEGA [97].

Results
A total of 126 Gyrodactylus specimens were found to parasitize 124 cypriniform fish host specimens belonging to 17 species, including two catostomid and 15 leuciscid representatives (Table 1).A total of 25 Gyrodactylus spp.were found, ten of them considered new to science and formally described below.Our investigation further revealed Gyrodactylus specimens representing eight potentially new species that have apparently never been described so far.Due to their small sample sizes, which preclude proper formal descriptions, these species are simply characterized based on the morphology of haptoral sclerites, and genetic information (when available).
Herein, Gyrodactylus specimens were firstly identified based on their haptoral sclerites and MCO when available.Overall, differential diagnosis involving congeners, mainly from Nearctic fauna, was provided for each identified species.Descriptions of new Gyrodactylus spp.(see below) were supplemented by genetic data according to the delineation within Gyrodactylus applied by Ziȩtara and Lumme [108] and Huyse et al. [53], with !1% of intraspecific genetic variation in the ITS region regarded as an upper limit.In total, 34 and 45 ITS and 18S rDNA sequences, respectively representing 22 Gyrodactylus spp.were successfully obtained.The size of raw fragments generated for each marker is included in the species description sections.nBLAST queries applied to ITS and 18S rDNA fragments (accessed in September 2022) revealed either no match or a few close hits with up to 100% similarity with already published sequences (Table 2).Sequences of the ITS regions showed higher intra-and inter-species genetic variation than 18S rDNA sequences which were highly conservative (Tables S1 and S2 in Supplementary material).

Differential diagnosis
So far, no formal descriptions of Gyrodactylus spp.parasitizing L. chrysocephalus or S. atromaculatus are available [11].When comparing G. hanseni n. sp.(Figs.1C and 1D) specimens from the two fish hosts, a weak variation is observed, mainly in (i) the ventral bar membrane, which has a broader ending in G. hanseni n. sp.from L. chrysocephalus compared to that from S. atromaculatus, (ii) the dorsal bar, which is slightly curved with an irregular wall in G. hanseni n. sp.from L. chrysocephalus, but well curved with posterior projections near each end in G. hanseni n. sp.from S. atromaculatus, and (iii) the shape of marginal hooks where the sickle foot in specimens from L. chrysocephalus lacks the shelf in the sickle toe, a feature present in specimens from S. atromaculatus.Regarding the Gyrodactylus sp. from captive N. crysoleucas, which is genetically identical to G. hanseni n. sp.(see below), Leis et al. [54] assumed this species (holotype of poor quality) to be G. variabilis Mizelle & Kritsky, 1967 [68] formally described from non-native N. crysoleucas (introduced in California, see [90]).The sizes of haptoral sclerites in specimens from L. chrysocephalus and S. atromaculatus considerably overlap with those in Gyrodactylus sp. from N. crysoleucas.When comparing our specimens to those of G. variabilis, considerable variation in the dorsal bar is observed (19.6-28.6 lm in G. hanseni n. sp. vs. 12-14 lm in G. variabilis).Leis et al. [54] reported further differences in the shape of the sickle of the marginal hooks (a more compact sickle in Gyrodactylus sp. from N. crysoleucas vs. a long and thin one in G. variabilis).Previous parasitological investigation of S. atromaculatus [11] in Eastern Canada revealed the presence of a single specimen of Gyrodactylus sp.So far, their study represents this host's sole record of Gyrodactylus spp., but no drawings or measurements were provided.Therefore, it is impossible to state whether or not the specimen recovered by Cone [11] represents G. hanseni n. sp.Overall, the morphology of the haptoral sclerites of G. hanseni n. sp., especially that of the ventral bar, is strongly reminiscent of that of G. asperus Rogers, 1967 parasitizing the rough shiner Notropis baileyi Suttkus & Raney, 1955 [85]

Differential diagnosis
Herein, N. hudsonius has been investigated for Gyrodactylus spp.for the first time.Morphologically, G. huyseae n. sp.specimens from L. chrysocephalus and those from N. hudsonius did not show any obvious variation in their haptoral sclerites.Gyrodactylus huyseae n. sp.(Figs.2A and 2B) can be compared to G. baeacanthus from the blacktail shiner Cyprinella venusta Girard, 1856 (Leuscicidae) [103] and the comely shiner N. amoenus [49], G. dechtiari [32], and G. laevisoides [46]  regarding the overall morphology of their haptoral sclerites.However, the new species differs from G. baeacanthus mainly by the shape of the dorsal bar (constricted at the midpoint in G. huyseae n. sp. vs. straight and vacuolated in G. baeacanthus).Gyrodactylus huyseae n. sp. is distinguishable from G. dechtiari regarding its shorter anchors (33.4-36.4lm in G. huyseae n. sp. vs. 45 lm in G. dechtiari).It is different from G. laevisoides by (i) its relatively longer marginal hooks (20.5-24.5 lm in G. huyseae n. sp. vs. 17-19 lm in G. laevisoides), and (ii) the differently shaped ventral bar membrane (constricted distally in G. huyseae n. sp. vs. rectangular and distally rounded in G. laevisoides (visible in the original drawing, but not mentioned in the species description)).

Molecular taxonomy
Fragments covering ITS1 (369 bp), 5.8S (157 bp), ITS2 (389 bp), and 18S rDNA (439 bp) were successfully sequenced for three parasite specimens from C. spadiceum inhabiting South-central localities (Arkansas, USA) (Table 1).No intraspecific variation was found.The nBLAST search did not reveal any hit close to G. lummei n. sp.(Table 2) with sequences of both 18S rDNA and ITS region.Based on the morphological evidence, Gyrodactylus sp. 1 "C.spadiceum" was shown to be the closest congener to G. lummei n. sp.within our Gyrodactylus dataset based on the 18S rDNA sequences (p-distances = 1.2%, 5 bp; Table S2), a result not obtained with sequences of the ITS regions.

Differential diagnosis
No morphological variation was observed for G. prikrylovae n. sp.(Fig. 3B) on the geographical scale.Despite the high morphological similarity with G. scholzi n. sp.(see below), consistent differences in haptoral sclerites were found to support the distinction between these two species.These differences are as follows: (i) in the shape of the ventral bar membrane, which presents a knob in G. prikrylovae n. sp., and (ii) in the dorsal bar, which is very often curved, and constricted at the midpoint with posteriorly directed projections in G. prikrylovae n. sp., but mostly straight in G. scholzi n. sp.(see below).Since no morphology was included in [31], where a Gyrodactylus sp. that was genetically close to G. prikrylovae n. sp. was reported from the same host species, the newly described species is comparable on the basis of haptoral sclerites to G. hoffmani Wellborn & Rogers, 1967, a species widely distributed on P. promelas [40,68,69,103], and G. lacustris Mizelle & Kritsky, 1967 parasitizing the same host [24,68].Considerable overlap in the metrics of sclerotized structures was found in G. prikrylovae n. sp. and G. hoffmani.Yet, these two species can be distinguished from each other regarding the shape of (i) the ventral bar membrane (tapering to a rounded edge posteriorly in G. prikrylovae n. sp. vs. an almost rectangular one with sides tapering slightly in G. hoffmani), and (ii) the marginal hooks (a pointed toe in G. prikrylovae n. sp. vs. a blunt toe in G. hoffmani).Gyrodactylus prikrylovae n. sp. is discriminated from G. lacustris in having (i) shorter anchors (49.4-54.1 lm in G. prikrylovae n. sp. vs. 64-73 lm in G. lacustris), and (ii) slightly shorter marginal hooks (23.8-28.5 lm in G. prikrylovae n. sp. vs. 32-34 lm in G. lacustris).

Molecular taxonomy
Fragments covering ITS1 (388 bp), 5.8S (157 bp), ITS2 (392 bp), and 18S rDNA (439 bp) were successfully sequenced for a single G. prikrylovae n. sp.specimen parasitizing P. promelas from each of Northeastern and South-central regions (New York and Arkansas, USA, respectively) (Table 1).nBLAST search using sequences of the ITS regions and 18S rDNA indicated Gyrodactylus sp.(AY099507) from P. promelas sampled in Idaho (USA) [31], and Gyrodactylus sp.(KT149284) from captive N. crysoleucas [54] as the closest hits to G. prikrylovae n. sp., respectively.It should be noted that the query coverage of the published ITS sequence AY099507 was only 46%, as the ITS1 part and a portion of 5.8S were missing (Table 2).Weak intraspecific variation was found in ITS sequences on the geographical scale (Table S1).With an intraspecific variation exceeding the limit value with sequences of the ITS region (p-distances = 0.9% 7 bp, 1.6-1.7%,15 bp; Table S1) and no genetic variation in 18S rDNA sequences (Table S2), G. scholzi n. sp.(see below) was recovered as the closest congener among the studied species.

Differential diagnosis
No interspecific morphological variation was observed within G. scholzi n. sp.(Fig. 3C) on the geographical scale.Comparison between G. scholzi n. sp. and its closely related G. prikrylovae n. sp. is detailed above.A few haptoral features supported the distinction between G. scholzi n. sp. and each of G. hoffmani and G. lacustris, both from P. promelas [68,103].The new species is mainly different from G. hoffmani by the knob in the median part of the ventral bar, a feature absent in the latter.Similarly, this structure discriminated G. scholzi n. sp.from G. lacustris, in addition to (i) shorter anchors (49.0-56.5 lm in G. scholzi n. sp. vs. 64-73 lm in G. lacustris), and (ii) slightly shorter marginal hooks (24.9-27.7 lm in G. scholzi n. sp. vs. 32-34 lm in G. lacustris).

Differential diagnosis
Gyrodactylus steineri n. sp.(Fig. 3D) was recognized as a new and first species parasitizing C. elongatus.This species was formally described herein based on pertinent haptoral morphology, especially the typical shape (large and pronounced) of ventral bar lateral processes.The haptoral morphology shown by G. steineri n. sp. is reminiscent of that of newly described G. huyseae n. sp. and G. mendeli n. sp.from distinct leuciscid hosts (see above), as well as that of G. asperus from N. baileyi [85] and G. parvicirrus from N. atherinoides [86].Gyrodactylus steineri n. sp. is distinguishable from G. huyseae n. sp. in having (i) longer anchors (63.1-67.7

New records for Nearctic Gyrodactylus species
Our study revealed the presence of seven Gyrodactylus spp.from two catostomid hosts and a single leuciscid host.Due to the small sample size for most species, no formal redescription is provided; we refer to these species as Gyrodactylus and characterized them with reference to DNA sequences (when available) and haptoral sclerites.

Differential diagnosis
The occurrence of G. atratuli on a range of Nearctic leusciscid hosts demonstrates its continentally wide geographic distribution and host specificity.In this study, we provided additional locality records to G. atratuli.Morphology of haptoral sclerites exhibited by our specimens of G. atratuli (Fig. 4A) and those described in [84] and identified in [32] is overall identical.Contrariwise, sizes of the sclerotized structures revealed slight intraspecific variation, mainly in (i) the anchors (58.2-64.4lm in this study vs. 66-68 lm in [32]), (ii) the dorsal bar (20.3-27.5 lm in this study vs. 17-19 lm in [84]), and (iii) the marginal hooks (28.8-35.5 lm in this study vs. 25-28 lm in [84]).Herein, R. atratulus hosted, in addition to G. atratuli, two other distinct species that remain undescribed for lack of sufficient material (see below).Morphologically, G. atratuli differs from Gyrodactylus sp. 1 "R.atratulus" by the absence of a knob in the ventral bar in the former species.Gyrodactylus atratuli is further distinguishable from Gyrodactylus sp. 2 "R.atratulus" in having (i) a shorter ventral bar membrane (16.1-22.1 lm in G. atratuli vs. 37.8 lm in Gyrodactylus sp. 2 "R.atratulus"), and (ii) the lack of a filament in the handle of the marginal hooks in G. atratuli.

Differential diagnosis
Morphologically, our specimens representing G. colemanensis (Fig. 4B) and those described by [68] overlapped considerably in terms of metrics and the shapes of hard parts.Our study can thus be considered the first one reporting the presence of G. colemanensis on E. maxillingua in Northeastern watersheds.

Differential diagnosis
Gyrodactylus dechtiari (Fig. 4C) is known from widely distributed Rhinichthis species in the Nearctic region (see previous records above).This study thus presents R. cataractae from Northeastern localities in the USA as a new habitat record for G. dechtiari.The first description of G. dechtiari was very brief and included a limited number of measurements and a comparison with a few, though not very morphologically similar, congeners parasitizing unrelated fish hosts [32].Previous records of G. dechtiari [22,23] did not include any morphometric characterization.Despite the small sample size herein, more detailed haptoral morphology is provided (see above).Overall, the shape and size of the haptoral sclerites exhibited by the collected specimen identified as G. dechtiari are identical to those included in the original description [32].The only exception seems to be the dorsal bar, which is slightly longer in our specimen (18.6 lm vs. 13 lm in [32]), but this remains to be verified by the investigation of more specimens in the future.
Present study: C. commersonii, Rom Hill Beaver Pond, Cooperstown, and Leatherstocking Creek, Otsego, both in New York, USA Site of infection: fins.

Differential diagnosis
The haptoral morphology exhibited by G. spathulatus (Fig. 4D) in our study was in accordance with that in the original description of Mueller [71].This provides a new locality for G. spathulatus parasitizing Northeastern C. commersonii.Since the morphology of G. spathulatus was presented only in [71] (no morphological characterization of sclerotized structures was provided in [31]), our specimens are compared with those of [71].Although Mueller [71] provided clear drawings, he supplemented them with very limited measurements of the haptoral sclerites, including the lengths of the anchors and marginal hooks only.We added the above detailed measurements for G. spathulatus.Overall, the haptoral sclerites of the examined specimens exhibit similar shapes regarding the sclerotized structures when compared to those of specimens included in [71].The only difference is in terms of size, the parasite anchors in this study appear shorter than those in [71] (106.1 lm vs. 120 lm, respectively).
Fragments covering ITS1 (369 bp), 5.8S (157 bp), ITS2 (396 bp), and 18S rDNA (439 bp) were successfully sequenced for a single G. spathulatus specimen from Northeastern C. commersonii (New York, USA) (Table 1).nBLAST search based on the ITS and 18S rDNA sequences revealed high similarity, but with weak variation, between our specimen and already published G. spathulatus (JF836152 and JF836098) [31].It should be noted that the coverage of the published ITS sequence was only 46%, as the ITS1 part and a portion of 5.8S were not previously sequenced (Table 2).Thus, the published ITS sequence for G. spathulatus was not included in the genetic variation calculation (Table S1).Based on the ITS regions and 18S rDNA sequences, G. stunkardi Kritsky & Mizelle, 1968, known from a range of distant hosts (see below), appeared genetically the closest to G. spathulatus, yet with sufficient variation (Table S1 and S2).

Differential diagnosis
The shape of haptoral sclerites exhibited by our specimens of G. stunkardi (Fig. 5A) overlapped with that of those described by [49] and no particular variation was observed.The present study thus extends the geographical distributional range of G. stunkardi on the continental scale.

Differential diagnosis
Gyrodactylus variabilis (Fig. 5B) is already known from N. crysoleucas, but from Western localities in the USA, and on the same host in California, which represents an alien fish in this region [68].This means that our study extends the geographical range of G. variabilis to Northeastern and South-central USA and Canada.Regardless of the sample size, the metrics of the haptoral sclerites, mainly the anchors, in specimens sampled in USA were closer to those obtained from specimens of the original description of G. variabilis [68] than to those obtained herein from Canadian fish hosts.Additionally to G. variabilis, N. crysoleucas is known to host G. crysoleucas Mizelle and Kritsky, 1967 [68], G. rachelae Price and McMahon, 1967 [81] and G. wellborni Nowlin, 1968 [74].Previously, many Gyrodactylus spp.were recognized as a cause of gyrodactylosis in N. crysoleucas farms, but none of these species, including G. variabilis, was recognized since they were mostly misidentified at that time (see, for instance, [92]).Later on, there were a few records of Gyrodactylus sp. on wild-caught N. crysoleucas occurring in various freshwater habitats in Ontario [21,31], and Nova Scotia [27] (both in Canada).These field studies did not investigate the parasite haptoral morphology, which makes it hard to know whether G. variabilis was one of the collected species.

Differential diagnosis
Overall, G. wardi (Fig. 5C) specimens studied herein exhibited similar haptoral morphology to that of previous records, all collected from a range of Catostomus spp.Yet, the sclerotized structures were shown to vary slightly at host species level (see measurements in [49,66,99]).The haptoral morphology exhibited by G. wardi is reminiscent of that of the newly described G. hamdii n. sp.The main differences in the sclerotized structures that allow these two species to be distinguished are in the size of anchors and ventral bars (see above in the differential diagnosis of G. hamdii n. sp.).
Fragments covering ITS1 (415 bp), 5.8S (157 bp), ITS2 (389 bp), and 18S rDNA (434 bp) were successfully sequenced for a single G. wardi specimen from Northeastern of C. Catostomus (Quebec, Canada) (Table 1).nBLAST search did not reveal any close hit to G. wardi (Table 2), while G. hamdii n. sp. was again shown to be the closest congener based on the ITS region (see above).

Discussion
The investigation of viviparous gyrodactylids (Gyrodactylidae) parasitizing cypriniform fish hosts from distant Nearctic watersheds provided a good opportunity to assess the morphological and genetic diversity of these ectoparasites on a large continental scale.So far, gyrodactylids are the second largest monogenean family parasitizing Nearctic cypriniforms, with 54 Gyrodactylus spp.[51].Our survey focused on a range of cypriniform fish species, including 15 and two species of Leuciscidae and Catostomidae, respectively collected from various lakes and rivers associated with distinct drainage systems in the USA and Canada.Overall, 25 Gyrodactylus spp.were identified, of which 18 have never previously been described.A total of 10 species were newly described herein based on a combination of morphological traits (haptoral sclerites) and genetics (sequences of the ITS regions and 18S rDNA) except for G. steineri n. sp.(see above).Two Gyrodactylus spp., specifically G. ellae n. sp. and G. hamdii n. sp., were described from the widely distributed catostomid C. commersonii.Similarly, two new Gyrodactylus spp.were described from each of the following two leuciscid species, specifically G. mendeli n. sp. and G. steineri n. sp.from N. biguttatus, in addition to G. prikrylovae n. sp. and G. scholzi n. sp.from P. promelas.Two other Gyrodactylus spp.were described, each from a single leuciscid speciesspecifically, G. kuchtai n. sp.from C. neogaeus and G. lummei n. sp.from C. spadiceum.Finally, two other species were described, each found on two leuciscid speciesspecifically, G. hanseni n. sp.from L. chrysocephalus and S. atromaculatus, and G. huyseae n. sp.from L. chrysocephalus and N. hudsonius.The remaining eight potentially new species were morphologically and genetically (when DNA sequences were available) characterized to provide background for further investigations when additional samples are obtained.Presently, insufficient sample sizes with respect to these parasites preclude accurate species descriptions.They concern two undescribed species found to parasitize C. spadiceum, a single species from each of C. neogaeus, C. venusta, the Mississippi silvery minnow Hybognathus nuchalis Agassiz, 1855 and Lythrurus sp., and finally three undescribed species hosted by R. atratulus (see below).
Combining morphological and genetic analyses has nowadays become a common practice in monogenean species identification [5].While both analyses individually show specific limitations, together they provide more accurate taxonomic support for Gyrodactylus spp.[55].In this group, the morphological features used for species description are related almost exclusively to the shape, size, and proportions of several haptoral structures [59].Considering the high species richness of Gyrodactylus [2], non-significant morphological variations are expected, these causing insoluble confusions in species distinction [26].DNA segments such as the ITS regions and, to a lesser degree, the 18S rDNA have been shown to be successful markers for revealing new species, and for assessing intraspecific variability [18,76,110].
In accordance with Ziętara et al. [110], we confirmed the utility of each part of the ITS regions in Gyrodactylus spp.delimitation.It should be noted that, in this study, some Gyrodactylus specimens were not subjected to both morphological and genetic analyses because of the limited sample size or unsuccessful sequencing.Moreover, morphological differences in the attachment organ are essential components of accurate species delimitation in Gyrodactylus [109].Controversial taxonomy is mainly related to high levels of morphological intraspecific variation and interspecific similarities.Ziętara and Lumme [109] suggested that species delimitation is closely related to host specificity.In our study, G. hanseni n. sp. was found on L. chrysocephalus and S. atromaculatus, both inhabiting Midwestern localities.The weak sample size of G. hanseni n. sp.specimens parasitizing the latter host could suggest an accidental infection during the manipulation of the fish.However, a disparity was noticed in the haptoral morphology of G. hanseni n. sp.specimens in two host species, while although weak, a genetic variation was recovered.This may be connected with the fact that variation in haptoral sclerites may facilitate the colonization of new host species without corresponding genetic diversification, which is partly in accordance with a study on monogenean communities parasitizing marine Sparidae [44].The presence of G. hanseni n. sp. on unrelated fish hosts, but yet occurring in overlapping habitats, makes it a generalist species that was probably host-switched to additional host species from the main host species [95].Larger sample sizes of this Gyrodactylus species, covering a wider distributional range of the hosts, and subsequent host-parasite cophylogenetic analyses potentially feasible in the future will help in revealing the scenario of Gyrodactylus diversification.
Results obtained for G. huyseae n. sp.from L. chrysocephalus and N. hudsonius were ambiguous.Indeed, specimens of G. huyseae n. sp.overlap in each of their host geographical range [77], haptoral morphology (no consistent shape/size variation), and preferences for the site of infection (infect the fins in both cases).Contrariwise, genetic data obtained for G. huyseae n. sp. are questionable with respect to the variation in the sequences of each of ITS regions where the genetic variation slightly exceeded the limit value, and in that of the 18S rDNA where a single mutation was present.Genetic distances higher than 1% could indicate interspecific differentiation when these differences are accompanied by a meaningful ecological pattern [108].For distinct systems, different limiting values for intraand interspecific variation based on the ITS sequences were observed; i.e., 1.14% for African Gyrodactylus spp.[89], while up to 5% was retained for Neotropical communities [96].Considering our results for G. huyseae n. sp., two scenarios are possible.The first one is that the specimens parasitizing L. chrysocephalus and N. hudsonius represent two distinct species.The weak sample size did not allow us to safely discriminate specimens of two potential species and elaborate two distinct formal descriptions.Nevertheless, in this case the mismatches defined on morphology and on molecular genetics may reflect a complex speciation process of diversification involving a recent/ongoing gene flow.The second scenario is that G. huyseae n. sp. is a cryptic generalist species parasitizing unrelated C. Rahmouni et al.: Parasite 2023, 30, 40 but geographically overlapping fish hosts.The shared evolutionary history of the two leuciscid representatives may have played a role in sharing the same Gyrodactylus spp.Indeed, Luxilus has long been considered a subgenus of Notropis until Mayden [62] elevated the former to genus level.Moreover, hybridization may easily occur between L. chrysocephalus and N. hudsonius due to overlapping habitats for spawning [72].Revision of the taxonomic status of G. huyseae n. sp.using a higher sample size is suggested.Further, the use of mitochondrial markers like COI and microsatellites would detect potential introgression between parasite populations.
Our results revealed the complete conservation of 18S rDNA sequences among pairwise G. colemanensis; G. hanseni n. sp.; the unidentified Gyrodactylus sp. 1 "R.atratulus" and Gyrodactylus sp. 2 "R.atratulus"; and the previously published Gyrodactylus sp.(KT149284), all so far found to parasitize distinct cypriniform fish families (see above).This result was supported by haptoral morphology, but we revealed variations in the ITS sequences.Gilmore et al. [31] reported high similarity in 18S rDNA sequences; however, they considered this gene useful for the taxonomy and phylogeny of Nearctic Gyrodactylus spp.Our results based on partial sequences of this gene demonstrate that genetic convergence considerably reduces species-level resolution.We showed that some published 18S rDNA sequences of Gyrodactylus spp.fully matched newly generated sequences representing morphologically distinguishable species, and that ITS regions are an accurate tool for species delimitation.
In two distinct areas of the Nearctic region, each of two populations of P. promelas harbored one of the two newly described Gyrodactylus specimens, G. prikrylovae n. sp. or G. scholzi n. sp.As already observed for several species (see above), genetic variation based on their ITS sequences was around the limiting value for species delimitation, whilst their 18S sequences were identical.Gyrodactylus prikrylovae n. sp. and G. scholzi n. sp. were confidently separated according to differential morphological features related to the shapes of the dorsal and ventral bars and ITS sequences where 1.6-1.7%(15 bp out of 1016 bp) of inter-species genetic variation was found.Moreover, the nBLAST search using ITS sequences (although with weak coverage) showed that Gyrodactylus sp.(AY099507) from Northwestern P. promelas [31] was genetically closer to G. prikrylovae n. sp.than to G. scholzi n. sp.However, at this stage, it remains difficult to accurately assign Gyrodactylus sp. to one of these newly described species.
Our study revealed some morphological features typical for Nearctic Gyrodactylus lineages.This was the case with the knob observed on the ventral bar of G. scholzi n. sp., G. lummei n. sp., and G. stunkardi, a feature already reported together with a few other haptoral traits in Gyrodactylus spp. in this region [55].Investigating the phylogenetic relationships among Gyrodactylus spp.from different geographical regions and mapping their haptoral morphology onto phylogeny would reveal whether such specific characters potentially delimit some of the Nearctic lineages.Interestingly, two species parasitizing C. commersonii from closely North-eastern streams, namely G. ellae n. sp. and G. hamdii n. sp., showed distinct morphotypes.In addition, morphometric data regarding anchors, the ventral bar, and marginal hooks indicated sufficient evidence to support the identification of G. hamdii n. sp. and G. commersoni [99], both from distant C. commersonii populations, as two valid species.When considering ventral bar features like the absence of lateral processes and the spine-like shape of the membrane in G. ellae n. sp., which resembles the Palearctic G. elegans haptoral group [59], G. ellae n. sp. and G. hamdii n. sp. are highly distinguishable on the basis of morphology, which is in accordance with the variability in their ITS and 18S rDNA sequences.Furthermore, the haptoral morphology exhibited by G. ellae n. sp.highly resembles that of G. kuchtai n. sp. and the undescribed Gyrodactylus sp."C.neogaeus", both parasitizing phylogenetically distant cypriniform hosts.Their morphological similarity was in line with the pattern observed for DNA sequences, which showed relatively weak variation.These observations imply that Gyrodactylus spp.parasitizing distinct hosts are more closely related to each other than species occurring on the same host, which was the case of Eurasian Gyrodactylus of gobies [42].For other morphological traits, the marginal hooks exhibited by G. kuchtai n. sp. and Gyrodactylus sp. from C. neogaeus seem to be very similar to those of many representatives from Phoxinus spp.[2,83], which may mirror phylogenetical and historical relationships between European and Nearctic minnows.An exhaustive molecular study involving a larger sample size of Gyrodactylus spp., supplemented by the mapping of haptoral morphology onto parasite phylogeny, would contribute to the tracking of the morphological evolution and phylogenetic origin of Nearctic Gyrodactylus, its diversification on the continental scale, and its ancient biogeographical contacts with Eurasian congeners.
Finally, new data are presented in this study of the Nearctic Gyrodactylus fauna.Due to a lack of sufficient mounted specimens representing some species that may be potentially new to science, we present a brief morphological characterization, supplemented by genetic information (when available), for eight species without any formal description.Concerning previous records of Gyrodactylus spp. in the Nearctic region, we document seven species and present their haptoral morphology supplemented by genetic characterization.We report the presence of G. atratuli, G. stunkardi, and G. dechtiari, all previously known from leuciscid hosts with wide distributional ranges in the Nearctic area [22,23,32], and G. spathulatus and G. wardi mostly on widely-dispersed Catostomus spp.[20-25, 39, 49, 66, 71, 99].For G. atratuli collected from two distant localities, we revealed morphological intraspecific variation likely related to isolation-by-distance.As already suggested above, the presence of Gyrodactylus spp. on non-congeneric hosts could be the result of host switching.This scenario remains valid for the generalist G. stunkardi found to parasitize R. atratulis in this study, and previously identified mainly on congeneric [22,32], and rarely on geographically and phylogenetically distant catostomid [49], as well as on perciform fish hosts [23].It should be noted that C. occidentalis is restricted to Californian freshwater systems, which could identify G. stunkardi as an alien parasite in the western USA.Another Gyrodactylus spp.documented in our study was G. colemanensis.Morphology and genetics supported its presence in a Northeastern E. maxillingua population.Surprisingly, this parasite species was so far restricted to Nearctic captive salmonid hosts, which makes our findings involving representatives of wild leuciscid fish unique.This could be the consequence of ecological host switching followed by fish translocation for farming purposes, since the geographical distribution of salmonids overlaps with that of E. maxillingua [77].As suggested by Leis et al. [54], the newly generated ITS sequences confirmed the identity of Gyrodactylus sp.(KT149288) as G. variabilis, a species previously recorded from captive N. crysoleucas and originally described on the same host introduced to California.This study also allowed us to identify this species on N. crysoleucas across a wide native distributional range (USA and Canada).As obtained for G. hanseni n. sp., G. variabilis in our study showed variations in the ITS sequences around the limiting value for species delimitation.The host range recorded so far for G. hanseni n. sp. and G. variabilis and their morphological features, however, support species status for these species.

Table 1 .
List of cypriniform species investigated in the present study, grouped by host suborders, sample size, total body length, and localities of sampling in the Nearctic region, and list of Gyrodactylus species identified in fish hosts.USA: United States of America; CA: Canada.

Table 2 .
Summary of the nBLAST search for representative sequences of ITS regions (in bold) and 18S rDNA for available Gyrodactylus species related to Gyrodactylus species reported in the present study.Gyrodactylus species lacking DNA sequences or hits below 98% identity are not shown.For all species, the E-value was 0.0.GB AN: GenBank accession number.